1. Guidelines for Field Veterinarians and Para-professionals on the Surveillance of Avian Influenza KOIKE I. (Technical Consultant) 2007

2. 1.Review of Fundamental Information 2.NAI Surveillance Programme and Role of Field Veterinarians (FV) and Para-professionals (PP) 3.Surveillance Strategies: Important Roles of FV and PP for Clinical Surveillance, Serological Surveillance and Virological Surveillance 4.Serological and Virological Surveillance in Vaccinated Populations (Meaning of Sentinel Birds) 5.Endeavour Toward NAI Free Country/Province in Poultry; Case-Study in Some Country The Surveillance of Avian Influenza

3. 1. Review of Fundamental Information (1) Surveillance: ? The investigation Population Subpopulation Detect a pathogen agent / disease frequency & type (clinical, serological, virological) Epidemiology Purpose Desired output Ref.(1)OIE Terrestrial Animal Health Code(TAHC)– 2007, General Definition

4. 1. Review of Fundamental Information (2) Monitoring : ? The continuous investigation Population Subpopulation Environment changes in the prevalence of a disease/ characteristics of a pathogenic agent. Purpose Detect Ref.(1) OIE TAHC – 2007, General Definition

5. 1. Review of Fundamental Information (3) No Country Can Declare Itself Free From Avian Influenza In Wild Birds Ref.(2) OIE TAHC – 2007, Guidelines for the Surveillance of Avian Influenza (GSAI)

8. 1. Review of Fundamental Information (4) NAI ?:One of Notifiable Diseases: listed by the Veterinary Administration Detected, Suspected in the Field Ref.(1) OIE TAHC – 2007, General Definition, (4) OIE TAHC – 2007, Avian Influenza V Veterinary Authority Informing (National Regulation) NAI: Notifiable Avian Influenza Influenza A virus H5 subtype H7 subtype Any AI virus (IVPI>1.2 or >75%mortality)

9. 1. Review of Fundamental Information (5) H5, H7?: Hemagglutinin (HA:antigenic glycoprotein) on the surface of the influenza virus Ref: (3) Science 21-April 2006 Vol.312, (5) Wikipedia; Hemagglutinin, Neuraminidase, Hemagglutinin:16 different antigens at least, Subtypes: H1, H2,H3,H4,H5,H6 H7, H8,H9,H10,H11,H12,H13, H14, H15, H16 (1.biding to the receptors on the target vertebrate cells,. 2. causing the fusion of host endosomal membrane with the viral one) Neuraminidase: antigenic glycoprotein (enzyme) on the surface of the influenza virus, Subtype:N1,N2,N3,N4,N5,N6,N7,N8,N9 1. promoting the release of progeny viruses from infected cells, 2. preventing aggregation of viruses, 3. help the virus to enter the host by cleaving sialic acid from the host glycoprotein

10. 1. Review of Fundamental Information (5) Mechanism of Hemagglutinin Function Receptor Ref: (6) RCSB Protein Data Bank,

11. 1. Review of Fundamental Information (6) NAI HPNAI: Highly Pathogenic Notifiable Avian Influenza LPNAI: Low Pathogenicity Notifiable Avian Influenza 1. Intravenous inoculation (IC) (0.2ml,1/10 dilution of infective A.F ) susceptible 4 to 8 w.o (lethal at least for six of eight:75%)<10 days Or 2. Intravenous pathogenicity index (IVPI): (0.1ml, 1/10 dilution of A.F.HA>1/16) SPF, SAN 6 w.o. (1.2<mean score per bird per observation 24-h,10d) HPNAI Ref. (3) OIE TAHC – 2007, Avian Influenza, (7)OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (MDTVTA) NB: 3. H5, H7 : IC mortality test <75 %, IVPI < 1.2 Amino acid sequence at cleavage site of the hemagglutinin to be examined A.F: allantoic fluid, Result (H5 & H7 subtype except HPNAI) HPNAI Amino acid motif: non similar to other HPNAI isolates LPNAI Amino acid motif: similar to other HPNAI isolates

12. 1. Review of Fundamental Information (7) Amino acid sequence at cleavage site: HA 1HA 2 Cleavage site Ref: (3) Science 21-April 2006 Vol.312, (8) Wikipedia, Genetic code,(9)T.Horimoto et al. HA - - -P Q R R R K T R / G - - - Alanine:A, Arginine: R, Asparagine: N, Aspartic acid: D, Cysteine: C, Glutamine: Q, Glutamic acid: E, Glycine: G, Histidine: H, Isoleucine : I, Leucine: L, Lysine: K, Methionine: M, Phenylalanine: F, Proline: P, Serine: S Threonine: T, Tryptophan: W, Tyrosine: Y, Valine: V, Abbreviation of amino acid HPNAI motif: X-X-R/K –X- R/K-R, or R-R/K- R-K-T- R, e.g. --E R S R R K K R / G--- --R E R R R K K R / G--- A/muscovy duck/Jakarta/Sum 106/2006 (H5N1) HPNAI A/goose/Cambodia/28/2004 (H5N1) HPNAI --P Q R K R K T R / G--- A/chicken/Queretaro/14588-19/1994(H5N2) HPNAI --N V P Q R E T R / G--- LPNAI A/chicken/Jalisco/14585-660/1994(H5N2) X: nonbasic residues

13. 1. Review of Fundamental Information(8) Ref. (10) Wikipedia, Influenza

14. 1. Review of Fundamental Information (9) Access to OIE Terrestrial Animal Health Code, Access toOIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, Through Web Site OIE Home Page http://www.oie.int OIE Publications 1 st 2 nd Online Publications  Terrestrial Code  Terrestrial Manual  Aquatic Code  Aquatic Manual  Disease Information  etc For Latest Information

15. 2. NAI Surveillance Programme and Role of Field Veterinarians (FV) and Para-professionals (PP) The Purpose of NAI Surveillance To Detect NAI in Poultry Ref.(2) OIE TAHC – 2007, GSAI farmer/workerFV, PP’s communication with farmers Veterinary Authority V Chief of village Sample collection To Show NAI Free Country etc. Laboratory Testing Early warning system

16. 2. NAI Surveillance Programme and Role of Field Veterinarians (FV) and Para-professionals (PP) Free In accordance with OIE TAHC – 2007, GSAI Susceptible poultry population (non-vaccinated, vaccinated) Preceding 12 months Absence of NAIV Infection HPNAI infection found in a previously free country/zone LPNAI infection found in a previously free country/zone In accordance with OIE TAHC – 2007, GSAI Susceptible poultry population 3 months after a stamping-out policy, disinfection of all affected establishments Absence of NAIV Infection Regain Susceptible poultry population 3 months after a stamping-out policy, disinfection of all affected establishments Absence of NAIV Infection Active surveillance: at least every 6 months Ref. (3) OIE TAHC – 2007, Avian Influenza, Active Passive

17. 3.Surveillance Strategies: Important Roles of FV and PP for Clinical Surveillance, Serological Surveillance and Virological Surveillance  Clinical Surveillance * HPNAI, LPNAI  Serological Surveillance *Detection of antibodies against NAIV  Virological Surveillance * Monitor at risk population * Confirm clinically suspect cases * Follow up positive serological results * Test daily mortality * Ensure early detection of infection in the face of vaccination or in establishment epidemiologically linked to an outbreak FV / PP Information through communication, Sample collection, Liaison between veterinary authority and farmers Ref.(2) OIE TAHC – 2007, GSAI

18. bacteria virus protozoa mold other parasites heating humidity ventilation premise temperature raining Major Factors Affecting Health of Fowls premise structure vectors feed mice etc. wild birds personnel drinking water litter lighting predators Clinical Surveillance

19. Mortality Economic Loss 100 % Understanding of Major Viral Diseases 50 Newcastle (VVND) Disease Virulent Infectious Bursal Disease Marek’s Disease (Loss of Chickens, Egg Production, Growth Rate, Feed Efficiency etc.) Fowl Pox M type I. B EDS Highly Pathogenic Notifiable Avian Influenza (HPNAI) Y C Adult Birds A. E I L TI L T Others Reo CA AE: Avian Encephalomyelitis Reo: Reovirus Infection ILT: Infectious Laryngotrachitis CA: Chicken Anemia EDS: Egg Drop Syndromes YC: Young Chickens Cases M. type: Mucous-membrane type LPAI: Low Pathogenicity A I (except H5 & H7) LPNAI: Low Pathogenicity Notifiable A I VV: Velogenic Viscerotropic (Asian type) ND, Mesogenic LPAI LPNAI H5 & H7 Mutation Clinical Surveillance

20. Development of AI Outbreaks in Mexico (1) Reference: (11)Laureano(CPA), (12)M.Garcia(SPRL) 1980-1981: field investigation by Dr.Lorenzo T. & Dr.Benzamine L. (AGP test all negative) 1993 : Mexico Central Region(6 states) (decreased egg production, respiratory disorder etc.) 1994 (suspected: ND, Coryza, IB, Swelling syndrome etc.) Mar-Aug : Campaign for strengthening biosecurity May : Mexico Central Region—AI virus isolation from broiler farms (AIV: H5N2, LPAI)

21. Development of AI Outbreaks in Mexico (2) 1994 15 th Dec:Puebla State (Tehuacan district; AIV isolation from layer farms) AIV (H5N2) of HPAI,(10-15% mortality in layer farms) 1995 1 st Jan : Queretaro State(Marques, Atongo town) AIV (H5N2) of HPAI from parents stock farms 1 st Feb: Vaccine introduced (homologous type:local isolate) HPAI : 9 farms infected, LPAI: 424 farms (HPAI mortality :20-30% in breeders, 50% in broilers) Amount of suffered birds: about 49 million ones, 1995 5 th Jun: Last case of HPAI 2000 Guatemala 2001 El Salvador Sporadic occurrence of LPAI 2006 12 States: free, 20 States: eradication stage, LPAI only Approximate No. of birds infected/culled so far: >1,000,000,000 in Mexico, Guatemala and El Salvador Reference: (11)Laureano(CPA), (13)I.Capud(OIE) (14)John Summers (PIC,Canada) LPNAI

22. Lessons from the NAI Outbreaks in Mexico 1. Less Attention to LPNAI (H5) in the First Half of 1990s 2. Less Seriousness to LPNAI Because of Little Manifestation and Little Economic Loss in Management and Public Society 3. Less Seriousness to LPNAI in All Poultry Sectors (Lack of Public Awareness) Particularly for Biosecurity and Workers’ Stance 4. Easily Spread from One Farm to Another Farm (State) 5. Passages of the AI Virus in the Poultry Flocks 6. Mutation from LPNAI to HPNAI in Some Poultry Flocks 7. Crisis in the Poultry Sectors and Public Society

23. Serological Surveillance Detection of Type A : Nucleoprotein/ Matrix (NP/M) ELISA test Agar gel immunodiffusion (AGID) test Detection of Subtype: HA, NA, Serum Neutralization (SN) HI test NI test(FPS:NG) SN test FV / PP Blood collection with filter paper strip(FPS) / syringe-needle FPS:NG, serum from FPS is not suitable for NI test because hemolytic phenomenon affects OD measure

24. Serological Surveillance for the Detection of Antibodies Against NAIV Positive reactors mean; Natural Infection with NAIV Vaccination against NAIV Maternal antibodies derived from a vaccinated or infected parent flock are usually found in the yolk and can persist in progeny for up to 4 weeks Positive result due to the lack of specificity of the test HI test:Reliable in Avian Species, but not in Mammals AGID: Reliable for Detection of NP/M Antibodies in Chickens and Turkeys, but not in Other Avian Species ELISA: (blocking) Detect NP/M Antibodies in All Avian Species SN: Preferred Test for Mammals and Some Avian Species Ref.(2) OIE TAHC – 2007, GSAI

25. No. of farms surveyed ( No. of chickens tested) No. of positive ｆ arms ( No. of positive chickens) No. of negative ｆ arms ( No. of negative chickens) + ratio in farms(%) ( + ratio in chickens CB22 (44)10 (17)12(27)45 (38) BS11(31)8(10)3(21)73 (32) BG3(17)3(6)0(11)100 (35) DJ12(21)1(1)11(20)8.3 (4.7) Farm Site Test method: ELISA test kit for Detection of Antibody to AI (Type: A) Matrix/Nucleoprotein antigens (made in USA) Survey Year: 2002 AI Serological Surveillance in Some Country Target: Village chickens of free range under extensive rearing Factors: Bringing Pathogens, Transmitting Factors,

26. 4. Serological and Virological Surveillance in Vaccinated Populations (Meaning of Sentinel Birds) 1. Inactivated whole AI virus Homologous NA to Field Virus Heterologous NA to the Field Virus Setting of Sentinel Birds: Check of Infection virus isolation, detection of virus specific genome material or proteins detection of antibody to the field virus NA virus isolation, detection of virus specific genome material or proteins 2. Hemagglutinin expression- based vaccine (recombinant vaccine-fowl pox base) detection of antibody to NP/M virus isolation, detection of virus specific genome material or proteins

27. Endeavour Toward NAI Free Country / Province: Case-Study in Some Country

28. Development of AI Outbreaks in Mexico Reference: ( 15 )Chang-Won Lee(SPRL) Affected: 13/32

29. Current Situation of AI Map in Mexico Reference:(11) Laureano(CPA) Year Outbreaks (Detection of LPNAI) 2000 3 2001 4 2002 15 2003 11 2004 9 2005 49 (without vaccine) Free: 12/32, Eradication stage: 20/32

30. Countermeasure against AI Year HPAI Countermeasure Vaccine (dose) １９９５ ＋ Pa, Pn, Qi, S, te, V 383.2 million １９９６ 0 Pa, Pn, Qi, S, te, V 331.3 million １９９７ 0 Qf, Te, M, Su, V, Z 240.8 million １９９８ 0 Qf, Te, M, Su, V, Z Unknown １９９９ 0 Qf, Te, M, Su, V, Z Unknown ２０００ 0 Qf, Te, M, Su, V, Z 86.91 million ２００１ 0 Qf, Te, M, Su, V, Z 87 million ２００ 2 0 Qf, Qi Te, M, Su, V,Z 260.242 million ２００３ 0 V, Su, M, S, Qi, Z, 281.494 million ２００４ ０ Su, M, S, Qi (Jalisco State case) Pa: control programme for some areas of the country only or for certain types of breeding only, Pn: control programme for the whole country, Qf: Quarantine, precautions at the border, Qi: Movement control inside the country, S: stamping out, Te: testing, V: vaccination, Su: Surveillance, M: monitoring, Z: Zoning, Reference:(11), (16)OIE, Handistatus II Multiannual disease status Nationwide

31. Countermeasure against AI 1. Inactive vaccine with oil adjuvant Seed virus: CK/Mexico/232/94(H5N2) controlled by CPA* (field isolate of LPAI) 2. Recombinant vaccine ( Vector: Fowl Pox) in Mexico -1. H5 hemagglutinin gene from AI virus A/turkey/Ireland/83, -2. Developed in the Lab. of Enzo Paoletti, Canada, during mid-1980’s (AI gene insert being provided by Robert G.Webster of St. Jude Children's Research Hospital) -3. Merial Co. purchased the vaccine and is licensed by USDA. -4. 850 million doses have been used in Mexico, Guatemala, and El Salvador since 1997 Reference:(11), (17)D. Swayne, *Comision Mexico-Estados Unidos para Prevencion de la Fiebre Aftosa y otras Enfermedades Exoticas en los Animals

32. Countermeasure against AI Vaccine maker Strain (H5N2) Product Laboratorio Avimex A/Chicken/Mexico/ 232/94/CPA * Avian Influenza (mono: inactive) *Newcastle-Influenza (duo: inactive) Boehringer Ingelheim A/Chicken/Mexico/ 232/94/CPA *Enza-vac (mono:inactive) * Enza-vac+NDV (duo:inactive) IASA(IASA- Biommune) A/Chicken/Mexico/ 232/94/CPA * Influenza Aviar-Newcastle (duo:inactive) IntervetA/Chicken/Mexico/ 232/94/CPA * Nobilis IA Inac (mono:inactive) * Nobilis IA+ND Inac(duo:inactive) * Nobilis IA Coryza Inac(duo:inactive)

33. HPAI Vaccine Strain HPAI Guatemala, El Salbador (12)M.Garcia, (15)Chang-Won Lee(SPRL) Antigen Drift

34. *Endeavor for Eradication of AI in Jalisco State *Diagnosis in The Laboratory J a l i s c o

35. Surveillance Procedure Kinds of BirdsSample Commercial birds (broiler, layer,etc) Living birds, Cloaca swabs, Trachea swabs, Organs, Feeder swabs, (trachea/cloaca swab:60 pcs/flock, whole body:15-20/flock) Back yard birdsCloaca swabs, Trachea swabs, Organs, (trachea/cloaca swab: all birds/flock, organs:case by case) Fighting cockCloaca swabs, Trachea swabs (trachea/cloaca swab:30 - 60/flock, OstrichCloaca swabs, Trachea swabs (trachea/cloaca swab:60 pcs /flock, ) Hobby/Pet, Others Cloaca swabs, Trachea swabs, Organs (trachea/cloaca swab: all birds/flock, organs:case by case)

36. Frequency of Surveillance Commercial Production Sector Broiler Industry Layer Industry Breeding Stock Farms for Broilers/Layers/Others Parents Stock Farms Frequency Inspection Fee Every time of marketing Payment by themselves At least 6 months Payment by themselves Backyard Raising/ Fighting Cock/ Pet Bird/Zoo Animal etc. At least 6 months Covered by Regional Livestock Committee

37. Virological Surveillance Trachea Lung Spleen Cerebrum The same organs of the same flock shall pool as one sample respectively Homogenized suspension of each sample shall inoculate into 5 CE eggs,9-10 d.o.,respectively. Incubate eggs in a hatcher for 4 – 5 days. Observe eggs every day. The same procedure as the above

38. Achievement of Monitoring from 2002 to 2005 Year Item 2002200320042005 No. of districts monitored to total districts 25 124 37 124 113 124 ? 124 Ratio (%) 20 2991? Administrative Districts Monitored Total birds inspected with virological surveillance per year: 15,000 on the average

40. Review of AI Virus(H5) Isolation through Monitoring from 2002 to 2005 in Jalisco State No.123456789101112131415 Year 0203 04 Farm ABBCCDDEFGHHIJK District TZES CC ZP TNEZ IXGFTI Plaque Test _______________ PCR* Matrix ＋ ＋＋＋＋＋＋＋＋＋＋＋＋＋＋ PCR* H5 ＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋ Type LP LP: low pathogenic, * Ref (17)Erica Spackman et al., All of positive farms were commercial broiler ones. Plaque Test: Chicken Embryo Fibroblast Culture without Trypsin

41. Surveillance Toward NAI Free Country/Province in Mexico Control stage: Epidemic, Stamping out/Vaccination etc. Strengthen Biosecurity, Eradication Strengthen Biosecurity stage : 1 St Sporadic, partly vaccination, 2 nd Rarely occur, vaccination prohibited No occurrence in the field during 1-2 years Free stage : No occurrence without vaccination under strengthened biosecurity Monitoring supported by field vet., para-prof. (Monitoring, Surveillance) Carry on Monitoring Reference: (18) NOM-044-1995, Surveillance: clinical, serological, virological (supported by field vet., para-prof.) (field vet., para-prof.) Monitoring

42. Others Aquatic migratory birds Reference: (19) Horimoto, (20) R.G. Webster

43. Lessons from the Countermeasures in Mexico 1. Adoption of Vaccination Partly success for control Antigen drift has taken place Less effect in vaccination 2. Thorough Serological Surveillance and Virological One 3. All Birds (Poultry & Pet Birds) Reared by Public The Targets of Surveillance 5. Vaccination Shall be Ceased in Accordance with the Progress of Control/Eradication Stage 4. Virological Positive Flock Stamping Out

44. Thank you very much October 2007

45. The Dawn Comes Surely

46. (1)OIE Terrestrial Animal Health Code(TAHC)– 2007, General Definition (2) OIE TAHC – 2007, Guidelines for the Surveillance of Avian Influenza (3) Science, Vol. 312, 2006 April 21, (4) OIE TAHC – 2007, Avian Influenza (5) Wikipedia; Hemagglutinin, Neuraminidase, http://en.wikipedia.org/wiki/Hemagglutinin, http://en.wikipedia.org/wiki/Neuraminidase,http://en.wikipedia.org/wiki/Neuraminidase (6) RCSB Protein Data Bank, http://www.pdb.org./pdb/static.do?p=education_discussion/molecule_of_the_month/p-- (7)OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, Avian Influenza (8) Wikipedia, Genetic code, http://en.wikipedia.org/wiki/Genetic code,http://en.wikipedia.org/wiki/Genetic (9) T.Horimoto, Y.Kawaoka, Journal of Virology, May,1994,p.3120-3218 (10) Wikipedia, Influenza, http://en.wikipedia.org/wiki/Influenzahttp://en.wikipedia.org/wiki/Influenza (11) Laureano Vazquez M., Panel Discussion about AI, 2005, 11,3, CUCBA, Guadalajara, Jalisco, Mexico (12) M.Garcia et al. Journal of General Virology (1996),Vol. 77,1493 – 1504, (13) I. Capud et al.; Currently available tools and strategies for emergency vaccination in case of avian influenza, http://library.wur.nl/frontis/avian_influenza/idex.html http://library.wur.nl/frontis/avian_influenza/idex.html

47. (15) Chan-Won Lee, et al., Journal of Virology(2004) Vol.78, No.15, 8372 – 8381, (16) OIE, Handistatus II Multiannual disease status, 2005 (17) D. Swayne et al. Av Dis. (1997) 41, 910 – 922, (18) Erica Spackman et al., Journal of Clinical Microbiology (2002) Vol.40, No.9, 3256-3260, (19) NOM-044-1995, Mexico (20) Horimoto et al. Virology (1995) Vol.213, 1, 223 – 230, (21) R.G. Webster, Influenza: An Emerging Disease, http://cdc.gov/ncidod/eid/vol4no3/webster.htm (14) John Summers, http://poultryindustrycouncil.ca/Factsheets/fact59.htmhttp://poultryindustrycouncil.ca/Factsheets/fact59.htm